Conformational Flexibility in DNA Nanoconstructs: A Time-Resolved

J. Phys. Chem. C 2008, 112, 13089–13094

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Conformational Flexibility in DNA Nanoconstructs: A Time-Resolved Fluorescence Resonance Energy Transfer Study Peter Sandin,* Per Lincoln, and Bo Albinsson Department of Chemical and Biological Engineering/Physical Chemistry, Chalmers UniVersity of Technology, SE-41296 Gothenburg, Sweden ReceiVed: February 29, 2008; ReVised Manuscript ReceiVed: May 15, 2008

Time-resolved fluorescence resonance energy transfer has been used to investigate the conformational flexibility of a DNA nanostructure. As the size of DNA nanostructures are becoming increasingly smaller, commonly used methods for characterization like atomic force microscopy (AFM) will become increasingly more difficult. Here we demonstrate that time-resolved fluorescence resonance energy transfer (tr-FRET) can serve as a tool for retrieving structural information on small DNA nanostructures. We have formed a DNA pseudohexagon using six different standard 22-mer oligonucleotides. The construct was labeled with a donor (fluorescein) in one corner while “walking” the acceptor (Cy3) from corner to corner around the hexagon. This resulted in five differently labeled constructs that were investigated with time-correlated single photon counting. The distribution of donor-acceptor distances present in each sample was estimated from analyzing the donor emission decays using a Gaussian distribution model. The results show a relatively wide distribution for all measured distances, fwhm between 14 and 33 Å, indicating that the pseudohexagonal motif is a flexible structure. In addition the recovered mean distances between the donor and acceptor follow the expected theoretical trend very well. The results show that, not only is tr-FRET an alternative method for retrieving structural information from DNA nanostructures whose size are below the present resolution of AFM but also, it demonstrate the potential of tr-FRET as a method for probing local flexibility in larger arrays of this kind. Introduction In the past decade, the field of DNA nanotechnology has grown significantly. Many different approaches to build and control the formation of DNA nanostructures have been employed. The pioneer in this field, Nadrian Seeman, has focused mainly on the use of linear oligonucleotides to form rigid crossover motifs that were made to form extended, repeating two-dimensional arrays.1–4 Other groups such as Turberfield et al. and Joyce et al. have shown that natural linear DNA can be designed to form also advanced three-dimensional structures such as a DNA tetrahedron5 and a DNA octahedron.6 Synthetic non-nucleic acids have also been incorporated into DNA to be used as linkers or branching points by, for example, von Kiedrowski et al.,7–9 Sawai et al.,10 and Aldaye and Sleiman.11–14 More examples of DNA-nanostructures and advances in the field have recently been reviewed.15–19 One goal in DNA nanotechnology is to build two and threedimensional arrays that have the potential use as rigid scaffolds for positioning molecular components with nanometer or subnanometer precision. The most common approach to this has been to form small rigid building blocks and then have them come together into large, rigid and well-defined DNA nanonetwork. However, there have also been other approaches in which monomeric units with flexible or semiflexible branching points have been used to produce well-defined repeating DNA arrays.3,20 The incorporation into larger constructs or arrays seems to induce order and rigidity into these flexible motifs. We recently found that a pseudohexagonal DNA nanostructure built using flexible building blocks was formed in very high * To whom correspondence should be addressed. Tel: +46 (0) 31 7723056. Fax: +46 (0) 31 7723858. Email: [email protected].

yields.21 A similar hexagonal structure, using rigid vertices as corners, was recently reported to be formed with a significantly lower yield and contaminated by higher order of polymeric species.11 Thus it seems that in this case, conformational flexibility is important for the formation of the monomeric motif. We recently proposed a new approach toward building an extended nonrepetitive two-dimensional DNA nanoarray using flexible pseudohexagonal structures as fundamental units.21,22 At the heart of this approach is a new synthetic three-way node. The node is fully compatible with standard automated DNA synthesizers and has orthogonal protecting groups on each arm which allows for full freedom of sequence, directionality, and DNA modifications of each individual arm.21 Using these threeway nodes we have successfully designed and formed both the hexagonal motif21 as well as the “dimeric” naphthalene-like motif.22 Since our structure, having sides of only ten base-pairs each, it is significantly smaller than previously published DNA nanostructures, the structural details are below the current resolution of AFM; the most commonly used technique for structural characterization in this field. In taking this step down in size and consequently not being able to rely on AFM for structural information, we entered into dimensions that are well suited for fluorescence resonance energy transfer (FRET). FRET is a tool for long-range distance determination, most sensitive in the range 10 to 80 Å. The measured efficiency of excitation energy transfer from a donor molecule to an acceptor depends inversely on the sixth power of the distance separating the donor and acceptor.23 FRET has successfully been used as a spectroscopic ruler in DNA research and, as such, provided much information on, for example, global DNA structures and DNA-macromolecular assemblies.24–30 However, the presence

10.1021/jp801790c CCC: $40.75  2008 American Chemical Society Published on Web 08/05/2008

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Figure 1. A color coded schematic drawing of the pseudohexagonal DNA structure to the left. To the right is a snapshot from the molecular dynamic simulation with the same color coding. Visible in the snapshot is also the fluorecein molecule attached at the 5′-terminus on strand one (yellow). The structure consists of six 22-mer oligonucleotides each having two different 10-mer outer regions complementary to 10-mer regions on two other 22-mer oligonucleotides and being orthogonal to any other part of all sequences. The two 10-mer regions are separated by two single stranded thymines (black) which serve as flexible hinges. Arrowheads indicate 3′-terminus, and blunt ends indicate the 5′terminus. All labels were attached to the 5′-terminus of the corresponding oligonucleotides positioning them at the corners of the construct (as indicated by the position of the numbers in the figure). The fluorecein donor label was always attached to strand 1 (yellow), and Cy3 acceptor labels were varied between all other 5′-terminus making up a series of five samples. Naming of the samples refers to the position of the Cy3 label, e.g., C3 is the Cyclized construct with donor at position 1 and Cy3 attached to strand 3 (green) positioning it at 3.

of multiple distances in a sample will make a quantitative data analysis of steady-state FRET significantly more complicated. When measuring on a dynamic system or multiconformational molecules where multiple distances are expected it is more useful to measure time-resolved FRET (tr-FRET). With trFRET, the distribution of distances between the donor and acceptor becomes available, and it has previously been used to study distance distribution in dye-linked oligonucleotides,31,32 DNA bending33 and conformational flexibility, and heterogeneity in helical DNA junctions and hairpin motifs.34–36 However, so far FRET has only been sparsely used in the field of DNA nanotechnology. This is most likely due to the fact that the size of most structures has so far been large enough for AFM and too large for FRET. In the present study, we have applied tr-FRET for investigating conformational flexibility and distance distribution in a previously reported pseudohexagon.21 Labeling one corner in the hexagonal structure with a fluorescein donor and varying the labeling position of the Cy3 acceptor gave us five different corner-to-corner distances measurable by tr-FRET. By analyzing the fluorescence decay of the donor for each sample we were able to estimate the distributions of these distances. The results show that the pseudohexagonal structure has relatively large conformational flexibility, as designed. Moreover, we demonstrate that tr-FRET shows promise to be a valuable tool for structural investigations of DNA nanostructures of this dimension. Material and Methods Formation of Fluorescently Labeled Pseudohexagons. All oligonucleotides, both labeled and nonlabeled were kindly provided by Professor Tom Brown (University of Southampton). The fluorescently labeled oligonucleotides were labeled in the 5′ end and connected by a 6 and 3 carbon long hydrocarbon linker for fluorescein and Cy3, respectively. For oligonucleotide sequences, see Supporting Information.

Sandin et al. Concentrations were estimated from the absorption at 260 nm. Three micromolar stock solutions of each unlabeled and Cy3 (acceptor) labeled strand were prepared together with a 1.5 µM stock solution of fluorescein (donor) labeled strand. The buffer used was a 50 mM Tris-HCl buffer with 500 mM NaCl at pH 8. Each sample was prepared by mixing 200 µL of the appropriate stock solutions and then annealed by heating the solution to 80 °C for 5 min and then a slow cooling over 8 h to 5.5 °C. After annealing, the samples were constantly kept at refrigerator temperatures (∼5 °C). The excess of acceptor labeled and unlabeled strands was to ensure that all donor strands were annealed into the final construct. The samples all have the fluorescein label attached to the same position while the Cy3 label is attached to a different corner in each sample (Figure 1). The different samples are referred to by the position of the Cy3 label, e.g., the cyclized sample with Cy3 in corner two is referred to as C2 (C stands for cyclized construct). Fluorescence Quantum Yield Determination. The measurements were performed on a SPEX fluorolog 3 spectrofluorimeter (JY Horiba) at 6 °C using an excitation wavelength of 490 nm. The fluorescence quantum yield (Φf) of the donor-only labeled cyclized construct was determined relative to the quantum yield of fluorescein (from Molecular Probes) in a 0.1 M NaOH solution at 25 °C (Φf ) 0.925).37 Fo¨rster Distance Determination. The rate of excitation energy transfer (FRET) between a donor and acceptor at a fixed distance, R, is given by the equation

( )

kT ) τD-1

R0 R

6

(1a)

where τD is the fluorescence lifetime in absence of the acceptor. The Fo¨rster distance, R0, was determined from the spectroscopic properties of the donor, fluorescein, and acceptor, Cy3. It was calculated using Equation 1b38

R60 )

9000(ln10)κ2ΦD 5

4

128π Nn

∫0∞ FD(λ)εA(λ)λ4 dλ

(1b)

where κ2 is the orientation factor describing the relative orientation of the donor- and acceptor-transition dipoles, ΦD is the fluorescence quantum yield of the donor in absence of the acceptor, N is Avogadro’s number, n is the refractive index of the medium, FD(λ) is the corrected fluorescence intensity of the donor in the wavelength range λ + ∆λ with the total intensity normalized to unity, and εA(λ) is the molar extinction coefficient of the acceptor at wavelength λ. The integral is the overlap integral and describes the spectral overlap between the donor emission and acceptor absorption. Time-Resolved Fluorescence Measurements. Fluorescence decays of donor-only and donor-acceptor labeled constructs were measured by time-correlated single photon counting. A Tsunami Ti:Sapphire laser (Spectra-Physics) pumped by a Millennia Pro X (Spectra-Physics) was used as excitation source. The pulsed output at 960 nm was frequency doubled to 480 nm and a pulse selector (Model 3980, Spectra-Physics) was used to reduce the repetition rate to 4 MHz before reaching the sample. The emitted photons were monitored at 520 nm and collected at magic angle conditions in a direction perpendicular to the excitation beam by a thermoelectrically cooled microchannel plate photomultiplier tube (MCP-PMT R3809U-50; Hamamatsu) and fed into a multichannel analyzer with 4096 channels (SPC-300, Edinburgh Analytical Instruments). A minimum of 10 000 counts were recorded in the top channel to achieve good statistics. The time-resolution in the measurement was 10-20 ps. All measurements were performed at 6 °C.

Conformational Flexibility in DNA Nanoconstructs

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Fluorescence Lifetime of Donor-Only Construct. The fluorescence decay curve of the donor-only construct was fitted to a two-exponential expression that was convoluted with the instrument response function using the software package FluoFit Pro v.4 (PicoQuant GMBH). Distance Distribution Calculations. The measured fluorescence decay curves for the donor–acceptor labeled constructs, IDA(t), were analyzed according to Equation 2

[

IDA(t) ) g(t) X (1 - f)

∫RR ∑ P(R)Ri max

min

i

]

exp × [(-t ⁄ τi)(1 + {R0 ⁄ R}6)]dR + fID(t) (2) where g(t) is the instrument response function measured on a silica sol scattering solution, X denotes convolution between the two functions, f is the fraction donor strand that does not undergo energy transfer, Rmin and Rmax are the minimum and maximum distance between the donor and acceptor, respectively, P(R) is the distance distribution function, Ri and τi are the intrinsic donor amplitudes and lifetimes and were obtained as describe above, R0 is the Fo¨rster distance, and ID(t) is the decay of donor in absence of the acceptor. For the distribution function, P(R), a one-dimensional Gaussian distribution was used

P(R) )

(

(R - Rmean)2 1 exp 2σ2 σ√2π

)

(3)

where σ2 is the variance and Rmean is the mean distance. The distribution was truncated at the extremes setting P(R) ) 0 where P(R) < 5 × 10-4. Equation 2 was fitted to the donor fluorescence decays by optimizing σ and Rmean in eq 3 and f. A multidimensional unconstrained nonlinear minimization method39 was implemented in the MATLAB 7 software package using a homemade program to optimize the fitting parameters. The goodness of fit was judged from the reduced χ-square value, χR2, and by examination of the weighted residuals. Molecular Dynamic Simulation. Geometry optimization and molecular dynamic simulations were made using molecular mechanics with an AMBER force field as implemented in the HyperChem 7.5 software package. Six identical oligonucleotide duplexes were joined together with single-stranded TT-links alternatingly in the 3′- or in the 5′-position to an approximate hexagon structure of 3-fold symmetry which was then energyminimized in vacuo. MD-simulations were run on the fluorescein labeled, cyclized construct, in vacuum at a constant temperature of 370 K, for 2000 ps with a 0.001 ps time step size. Distances were measured from atom number 9 in the xanthene ring system of the fluorescein moiety (see Figure S2 in Supporting Information) to carbon number two in either the purine or pyrimidine base on the nucleotide that the Cy3 was attached to. Distance data were collected from every 100th time step yielding a total of 20 000 distances collected for each of the five corner-tocorner distance. Results and Discussion Formation of Constructs. Since donor-acceptor distance distributions should be recovered from the fluorescence decays of the donor it is important to ensure that all donor-labeled strands are hybridized into the construct. Although previous results show that the cyclized construct forms in high yield, we used two equivalent excess of the unlabeled, as well as the acceptor-labeled strand to ensure that no single-stranded fluorescein labeled strands would be present. This was confirmed by gel electrophoresis in which no single-stranded fluorescein

labeled strands were detected (data not shown). However, small traces of fluorescein emission were detected from noncyclized structures in some samples. Although the intensity of these bands (when present) indicated this fraction to be very small, a parameter for possible unquenched fluorescein, i.e., the fraction of fluorescein not taking part of energy transfer to the acceptor, was allowed to be optimized in the fitting procedure. This parameter took a value of typically 0-1.1% confirming the gel electrophoresis result and showing that essentially all fluorescein labeled strands were found in the cyclized construct (Vide infra). Determination of the Fo¨rster Distance, R0, for the Fluorescein-Cy3 Donor-Acceptor Pair. The Fo¨rster distance, R0 was determined from the spectral properties of the donor-acceptor pair and calculated using eq 1b. The overlap integral, J(λ), was calculated numerically to 4.5 × 1015 M-1 cm-1(nm)4 and the donor fluorescence quantum yield, ΦD was measured to 0.66 when attached to the construct. These together with refractive index n ) 1.33 (the value in water assuming that dyes experience an aqueous environment) and using a dynamically averaged orientation parameter, κ2 ) 2/3, resulted in a R0 value of 61.6 Å. Different labeling positions showed no significant difference in calculated R0; therefore, 61.6 Å was used for all pairs. This is approximately 5 Å larger than previously reported R0 for this FRET pair.40,41 In comparison we have almost twice the donor quantum yield, which might stem from the environmental difference experienced by fluorescein at a corner of the pseudohexagon as compared to the environment when attached to the end of double-stranded oligonucleotides. It has previously been observed that the fluorescein emission is sensitive to what kind of construct it is attached to.32,35 In addition to this, we have measured the molar extinction coefficient of Cy3 attached to our construct to be 120 000 M-1 cm-1 which can be compared to previously published values of ∼90 000 M-1 cm-1.25,41 Lilley et al. have shown that when Cy3 is attached to the 5′-end of oligonucleotides, it stacks on the end of the helix40 which would most likely result in a hypochromic effect and thus lowering the molar extinction coefficient. In our case, with Cy3 being positioned at the corner of the pseudohexagon there is no blunt end to stack on since there are the two overhanging thymine bases. However, we do observe high fluorescence anisotropy (∼0.31) for Cy3 when attached to the construct indicating a strong dye-DNA interaction. Therefore, we conclude that although we have a strong interaction there is most likely less stacking than in pervious studies resulting in lesser hypochromic effect which in turn results in a higher extinction coefficient for Cy3 in our structure. The use of 2/3 as value for κ2 is motivated primarily by the low anisotropy of fluorescein,25,27 typically 0.1 with a slight dependence on labeled position as expected from the varying donor lifetime as a consequence of different energy transfer efficiencies. The influence from the orientation parameter was tested by analyzing the fluorescence decays with different fixed values of κ2. Within the limits of possible κ2 values, dictated by the degree of depolarization of the donor and acceptor,42–44 an expected shift in mean distances were observed but only marginal influence on the shape and width of the distance distribution was noted. It should be pointed out that a distribution of chromophore orientations at fixed donor-acceptor distance potentially could yield the same spread of energy transfer rates as a distribution in distances (cf. eqs 1a,b). This is a difficulty encountered in the application of FRET to structural determinations and it has been extensively discussed in the literature.24,33,34,42–48 However, in general including the κ2 distribution in the data analysis has only minor effect on the recovered donor-acceptor distance distributions.31,33–35 In our

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Sandin et al.

Figure 2. Examples of fluorescence decay curves for the fluorecein donor in donor-only construct (yellow) and donor-acceptor constructs C4 (blue) and C2 (red). Convention for naming the structures is found in Figure 1. Decays C4 and C2 are overlaid with the best fit using eqs 2 and 3 (solid black line). The instrument response is shown in orange. Samples were excited at 480 nm, and emission was collected at 520 nm. Measurements were performed at 6 °C.

constructs, based on structural considerations and molecular dynamics simulations (Vide infra), we believe that the distribution of distances dominates the observed distribution of fluorescence lifetimes but cannot rule out a small contribution from orientational distribution of the donor and acceptor. Fluorescence Decays of Donor-Only Construct. Lifetime analysis of the donor-only sample showed that the best fit was obtained by using two lifetimes. The major lifetime was 4.43 ns and the minor was 1.01 ns with preexponetial factors of 0.89 and 0.11, respectively. This shows that a small fraction of donors are quenched as a result from dye-DNA interactions in line with previous observations for other fluorescein labeled oligonucleotides.32,35 Corner-to-Corner Distance Distributions in the Pseudohexagon. Decays of the fluorescein emission were measured on five different samples, one for each corner-to-corner distance (Figure 1). In Figure 2, the measured fluorescence decays of sample C2 and C4 are shown as examples. These are the most and least quenched samples, respectively. Figure 2 also shows the fluorescence decay of the donor-only construct, which when compared to the two others clearly shows the fluorescence quenching effect of the acceptor. The decays were analyzed in terms of distance distributions using eqs 2 and 3 and recovered distributions and optimized parameter values are found in Figure 3 (upper panel) and Table 1, respectively. The distribution of distances for a labeled position is quite large as seen from the full width at half-maximum (fwhm) parameter that ranges between 14 and 33 Å. This indicates a high degree of conformational flexibility in the construct. It is also evident from the fwhm that the distributions become wider for longer distances. Comparing these results with the previously published results from steady-state FRET (Table 1), the mean distance in the distributions agrees very well with the trend found in the energy transfer efficiency.21 Also included in Table 1 are the calculated values for the fraction of donors not taking part in energy transfer and as can be seen the values are very small (0 - 0.011) indicating that virtually all fluorescein labeled strands are part of the cyclized constructs. For the longer distances (C3, C4, and C5), this parameter was zero. This however, does not necessarily mean that all fluorescein-labeled strands are hybridized into the constructs. In these positions, the distance between the donor and acceptor can be long enough

Figure 3. Normalized distance distributions for constructs C2 (red), C3 (green), C4 (blue), C5 (light blue), and C6 (violet). Upper panel shows the Gaussian distance distributions calculated from eq 3 using the best fit parameters recovered from the corresponding fluorescence decay curves. Lower panel shows the distance distributions retrieved from the molecular dynamic simulations.

TABLE 1: Donor-acceptor Distances and Recovered Fitting Parameters from Equations 2 and 3 constructsa

Essb

Rmean (Å)c

fwhm (Å)d

C2 C3 C4 C5 C6

0.81 0.40 0.30 0.32 0.66

46.4 68.9 72.7 70.7 50.9

13.9 26.5 32.9 24.3 20.0

f

e

0.006 0.000 0.000 0.000 0.011

χR2 1.27 1.24 1.12 1.24 1.26

a Convention for naming is found in Figure 1. b Steady-state energy transfer efficiencies obtained in ref. 21 c The mean distance in the recovered Gaussian distributions. d The full width of the distribution at half-maximum probability. e The fraction donor not taking part in energy transfer.

so that the donor will be unquenched even if it is part of the construct. Since there is no way to distinguish these two cases the small fraction of fluorescein in noncyclized constructs might be buried in the distribution. In all cases, the Gaussian distribution model gave excellent fits as seen from the χR2 values (χR2 < 1.27). It should however be mentioned that also other symmetric distribution models, for example an inverted parabola, gave equally good fits and with no significant difference in the recovered mean distances. Hence, the choice of distribution model had no significant effect on the result. It should also be pointed out that there was no need to scale R0 for the two different donor lifetimes as has previously been suggested,49 since the recovered structural parameters were essentially the same for both cases. To get an idea of how the pseudohexagon might behave in terms of dynamics, and to generate the possible distances we

Conformational Flexibility in DNA Nanoconstructs performed a rough qualitative molecular dynamic simulation in vacuo based on the AMBER force field on the donor labeled construct. We choose not to explicitly include the acceptor, Cy3, in the simulation since we were not sure of the exact position, only that it had strong interaction with the DNA. Instead we measured the distances between the fluorescein molecule and the last base pair on the double strand where Cy3 was attached. We did this in view of the study of Lilley et al., where it was suggested that Cy3 stacks on the blunt end of the oligonucleotide in which it is attached.40 In the simulation that ran over 2000 ps all five distances were collected every 0.1 ps resulting in 20 000 recorded values for each of the five different distances. These were plotted as statistical histograms shown in Figure 3 (lower panel). The result shows that there is a significant width of the distance distribution in all cases with the longer distances having the widest distributions, much like what was found in the experiments. In addition, the distributions seem relatively symmetric with a Gaussian shape. In a qualitative comparison between the simulation and experimental result we can see that the experiments show wider distributions and also the mean distances are slightly different. Wider distribution in the experiments can be expected since the simulation is performed in vacuum whereas the experiment is performed in aqueous buffer solution, which has a better ability to shield the electrostatic repulsion of the negative charges on the DNA allowing for more conformational flexibility. The difference in mean distances should not be overinterpreted since, as mentioned above, we use a crude approximation for the Cy3 position. Nevertheless, qualitatively, the two results agree very well. It should be pointed out that, when interpreting FRET results obtained using covalently linked dyes the possible contribution from the linker has to be considered. The linker will affect the distance but when measuring distance distributions it is more important to what extent the linker plus dye will affect the distributions. In this study, fluorescence anisotropy shows that the fluorescein moiety has a large rotational freedom suggesting that it extends out from the DNA and thus could potentially cause an apparent larger distance distribution even if the labeled object would be completely rigid. For this reason, apart from the distance distributions shown in Figure 3 an additional series of distance distributions were calculated from the simulation. The corresponding corner-to-corner distances were calculated using the phosphate to which the fluorescein was attached to so that contribution of the linker plus dye to the distributions could be evaluated. The result (see Supporting Information) was similar to the ones shown in Figure 3. The distribution was shifted about 5-10 Å, and the widths were in general slightly narrower, as could be expected. However, the major part of the distribution clearly reflects the flexibility of the construct and not the segmental flexibility of the linker plus dye moiety. An experimental measurement of the contribution of the linker to the distance distribution is difficult to obtain quantitatively. However, construct C2, in which the dyes are separated by a rigid ten base-pair long DNA duplex and no flexible TT hinges, allows us to get a qualitative estimate. Although simulations show that also two points at opposite ends of such a rigid duplex has a distribution of distances between them (see Supporting Information) the weight of the linker contribution to the total distance distribution is largest for this construct. Thus, even if the linkers alone are not responsible for the total width of the distribution, the C2 result may be viewed as an absolute maximum contribution of the linkers to the experimentally measured distance distributions. Comparing the fwhm for constructs C2-C6 (Table 1) shows that the fwhm of C2 is

J. Phys. Chem. C, Vol. 112, No. 34, 2008 13093 approximately half of that of C3-C6. This further supports the results from the simulations, namely that the major part of the distance distributions reflects the flexibility of the construct and not the segmental linker plus dye flexibility. Concluding Remarks We have used time-resolved FRET to show that the monomeric motif we intend to use as the building block for an extended nonrepeating DNA nanoarray has a relative large degree of conformational flexibility, as designed. Although the more common approach is to use rigid well-defined building blocks, we want to use the advantages of having flexible building blocks. We have previously shown that our structure is formed with very high yield, and it seems that the conformational flexibility may facilitate the formation. In an extended array, we expect that the flexibility of the hexagonal motif will decrease, as previously seen for other flexible motifs.3,20 In addition, the intended attachment of the array to a surface is also expected to decrease the flexibility. As the resolution of current AFM technology is not sufficient for imaging our structure it cannot provide the detailed structural information needed to give any insight into its conformational flexibility. Instead, this study shows that tr-FRET is capable of providing this information and reveals the conformational flexibility of the single pseudohexagonal motif. This also shows promise for tr-FRET as a method to retrieve structural details in arrays of this kind. Acknowledgment. We thank Professor Tom Brown (University of Southampton) for providing the oligonucleotides. This research is funded by the European Commission’s Sixth Framework Programme (Project Reference AMNA, Contract 013575). Supporting Information Available: Oligonucleotide sequences, structures of probes and additional simulation results. This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes (1) Li, X.; Yang, X.; Qi, J.; Seeman, N. C. J. Am. Chem. Soc. 1996, 118, 6131–6140. (2) Winfree, E.; Liu, F. R.; Wenzler, L. A.; Seeman, N. C. Nature 1998, 394, 539–544. (3) Mao, C.; Sun, W.; Seeman, N. C. J. Am. Chem. Soc. 1999, 121, 5437–5443. (4) LaBean, T. H.; Yan, H.; Kopatsch, J.; Liu, F.; Winfree, E.; Reif, J. H.; Seeman, N. C. J. Am. Chem. Soc. 2000, 122, 1848–1860. (5) Goodman, R. P.; Schaap, I. A. T.; Tardin, C. F.; Erben, C. M.; Berry, R. M.; Schmidt, C. F.; Turberfield, A. J. Science 2005, 310, 1661– 1665. (6) Shih, W. M.; Quispe, J. D.; Joyce, G. F. Nature 2004, 427, 618– 621. (7) Scheffler, M.; Dorenbeck, A.; Jordan, S.; Wustefeld, M.; von Kiedrowski, G. Angew. Chem., Int. Ed. 1999, 38, 3312–3315. (8) Eckardt, L. H.; Naumann, K.; Pankau, W. M.; Rein, M.; Schweitzer, M.; Windhab, N.; von Kiedrowski, G. Nature 2002, 420, 286–286. (9) von Kiedrowski, G.; Eckardt, L. H.; Naumann, K.; Pankau, W. M.; Reimold, M.; Rein, M. Pure Appl. Chem. 2003, 75, 609–619. (10) Kuroda, T.; Sakurai, Y.; Suzuki, Y.; Nakamura, A. O.; Kuwahara, M.; Ozaki, H.; Sawai, H. Chem. Asian J. 2006, 1, 575–580. (11) Aldaye, F. A.; Sleiman, H. F. Angew. Chem., Int. Ed. 2006, 45, 2204–2209. (12) Aldaye, F. A.; Sleiman, H. F. J. Am. Chem. Soc. 2007, 129, 4130– 4131. (13) Aldaye, F. A.; Sleiman, H. F. J. Am. Chem. Soc. 2007, 129, 10070– 10071. (14) Aldaye, F. A.; Sleiman, H. F. J. Am. Chem. Soc. 2007, 129, 13376– 13377. (15) Seeman, N. C. Mol. Biotechnol. 2007, 37, 246–257.

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